JP2007061028A - Functional food for ameliorating learning and memory disorder - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a functional food having excellent safety and containing an extract effective for the prevention or amelioration of learning and memory disorder, free from side actions and having medicinal effect and action favorably comparable with those of conventional medicinal pharmaceuticals. <P>SOLUTION: The functional food for ameliorating learning and memory disorder by the activation of CREB as an action mechanism contains crushed and squeezed juice of the whole fruit of citrus plant and/or an extract of the crushed and squeezed juice. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a functional food that improves learning and memory impairment, and more particularly to a functional food that improves learning and memory impairment using the activation of cAMP response element binding protein (CREB) as an action mechanism.

  In recent years, countermeasures for learning and memory disorders due to central nervous disease including dementia such as Alzheimer's disease have become one of the serious social problems, but the average life expectancy has been increasing year by year and the aging is rapidly progressing. In view of this, there is a need for an early solution of these problems.

On the other hand, as an important neuronal signaling pathway (brain information network) that controls and regulates learning memory, cyclic adenosine 3 ′, 5′-1 phosphate (cAMP) / cAMP-dependent protein kinase A (PKA) / The cAMP response element binding protein (CREB) system has recently become apparent. And it was confirmed that the learning and memory ability is enhanced when the signal transduction pathway in neurons is activated. Therefore, research and development of ethical drugs for activating CREB are being rapidly developed, and various therapeutic drugs have been published (see Patent Document 1).
JP-A-11-228417

However, the development of pharmaceuticals requires a long period of time and enormous costs, and there is an unavoidable risk that its use involves unpredictable side effects.
The present invention has been devised to solve such problems, and its main purpose is that it has no side effects to prevent or ameliorate learning and memory impairment, and compared with known medical drugs. Another object of the present invention is to provide a functional food excellent in safety, which contains an extract having a medicinal effect and action that is inferior.

  In order to solve such problems, the present invention contains at least one of a crushed juice of whole citrus fruits and an extract from the crushed juice, and uses CREB activation as a mechanism of action. We decided to provide a functional food that improves the characteristic learning memory impairment. In particular, the citrus fruits include hanayu, koraitachibana, shikitsu, pheasant, natsumikan, daidai, ponkan, mediterranean mandarin, dancy tangerine, giant beetle, tachibana, cobenimikan, nuclear-free kishu, shikwasa, ukulemikan, capuchi, ota ponkan, hirashu It may be selected from citrus fruits including sankitsu, cleopatra, koji, barley mandarin, and echan lemon.

  According to the present invention as described above, a great effect can be obtained in providing a functional food that can safely prevent and treat central nervous diseases including dementia such as Alzheimer's disease.

  Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

  The extract that exhibits the activation mechanism of CREB according to the present invention to improve learning and memory impairment is obtained from a certain kind of citrus fruits, and the citrus fruits are generally known to have a relatively strong astringency and sourness. Hanayu, Kourai Tachibana, Shikikitsu, pheasant, Natsumikan, Daidai, Ponkan, Mediterranean Mandarin, Dancy Tangerine, Obenimikan, Tachibana, Kobenimikan, Nucleus Kishu, Sikhwasa, Fukurimikan, Capphi, Ota Ponkan, Hiraukishu There are 24 species: Sankitsu, Cleopatra, Koji, Barbarian, and Yichang Lemon. Especially in Japan, Ponkan, Tachibana, Sikhwasa, and Eunshu are relatively easily available. Here, an example in Ponkan will be described.

  The whole fruit of Ponkan (fruit including the skin) is finely pulverized with a pulverizer, extracted three times with 80% ethanol water, and then the solvent is removed from the whole extract under reduced pressure to obtain a crude extract (extract ) For example, a mixture of 0.5% (V / V) Tween 80 and 0.5% (W / V) carboxymethyl cellulose (CMC) is added to this extract, or a vegetable oil such as corn oil is added to emulsify. And a homogeneous liquid was obtained by sonication.

  Sufficient medicinal effects can be obtained with this liquid, so it can be filled into soft capsules as capsules, or the liquid can be adsorbed onto an adsorbent powder and finely ground, then granulated and granulated. Or by tableting, it can be supplied in any dosage form for internal use as granules (bare tablets), as sugar-coated tablets, or by other known preparation techniques.

  Next, verification of the learning memory impairment improvement effect by the activation mechanism of CREB of the substance of the present invention will be described. In the verification, in vivo tests with small animals (pathological model animal tests for memory impairment) and in vitro tests (culture of rat hippocampal neurons; SDS-PAGE and Western plotting; plasmid introduction and reporter gene assay) It was decided to evaluate by.

[Creation of memory learning disorder model animals]
First, the in vivo test will be described.
Under anesthesia with an anesthetic drug Nembutal (50 mg / kg) administered intraperitoneally to a ddY male mouse weighing 24 to 26 g, a skull drilled into the skull above the olfactory bulb, and 2 / Three or more were removed by suction. This was sutured and sutured, the wound was smeared with a surgical adhesive, and an olfactory bulbectomy (Olfactory Bulbectomy, hereinafter abbreviated as OBX) mouse memory learning impairment model animal was prepared.
On the other hand, the group which did not perform a surgical operation was made into the control (Control) group.

[Measurement of memory learning behavior]
The behavior was evaluated using a step-through passive avoidance reaction device. Specifically, a mouse that enters the dark room after confirming that it has moved completely to the dark room by placing the mouse in the bright room side of the light / dark box where the two rooms, the light room and the dark room to which electrical stimulation is applied, is connected An electrical stimulus (1 mA, 500 msec) was applied to this, and this was used as a learning trial. Immediately after the completion of the learning trial, the above-described OBX treatment was performed and returned to the breeding cage. During the test, the latency time (reaction latency) is measured from the time it takes the mouse to enter the dark room until it enters the dark room, and this is used as a test trial. In this case, the mouse is electrically stimulated even if it enters the dark room. Did not give. In both of these learning trials and test trials, the cut-off time was set to 5 minutes, and mice that remained in the light room for 5 minutes or more in the learning trials were excluded from the test as ineligible individuals.

  In this measurement method, mice that have not suffered from memory impairment remain in the bright room for a long time because they retain memory of electrical stimulation as fear, but mice that have memory impairment retain fear. I often go into the darkroom because I am not. Using this difference, the response latency was used as an index of memory impairment.

[Verification of the improvement effect of the substance of the present invention on memory learning disorder model animals]
Comparison was made 7-10 times between the control group and the intraperitoneal administration (Extract) group of the whole fruit homogenate (510 mg / kg) as the test substance, the OBX group not administered with the test substance, and the control group for 11 days from 3 days after the OBX treatment. It was. A typical example is shown in FIG. However, only the solvent (mixture of 0.5% (V / V) Tween 80 and 0.5% (W / V) CMC) was intraperitoneally administered to the Control group.

  As a result, the response latency of the Control group was about 300 seconds (295 ± 5 seconds), whereas in the OBX group not administered with the test substance, it decreased to about 50 seconds (45 ± 11 seconds). Memory impairment was observed.

  On the other hand, in the Extract group to which the test substance was administered, the response latency was about 110 seconds (112 ± 15 seconds), confirming the clear effect of improving memory learning disorder.

  Next, the in vitro test will be described. It has been clarified that signals in nerve cells are transmitted from upstream to downstream by sequential phosphorylation of a series of proteins. CREB is activated when phosphorylated, binds to CRE causing gene switch-on, and transcription (expression) of the gene is initiated. By measuring the strength of transcription activity, the effect of the test substance can be increased. We decided to evaluate.

[Culture of primary rat hippocampal neurons]
As neurons for evaluation, hippocampal neurons were isolated from SD rats (E19; embryonic day 19 days, manufactured by Japan SLC). Specifically, the hippocampal region was cut out in a water-cooled phosphate buffer, and neurons were dispersed and purified using a SUMILON cell dispersion kit (manufactured by SUMITOMO BAKELITE CO., Ltd.). Dispersed and purified hippocampal neurons are suspended using Neurobasal Medium (1 × B-27 Supplement, 0.5 mM L-Glutamin, containing 0.005% Penicillin-Streptmycin) manufactured by GIBCO, and Poly-L Neurons were seeded at a cell number of 1 × 10 ⁶ cells / cm ² on a culture device coated with -lysine and cultured at 37 ° C. for 4 days in a 5% CO ₂ atmosphere. Thereafter, the medium was changed to Neurobasal Medium (containing 1 × B-27 Supplement, 0.5 mM L-Glutamin, 0.005% Penicillin-Streptmycin, 5 μM Ara-C), and the culture was continued. Thereafter, the medium was changed every 2-3 days.

[Verification of the enhancement effect of the substance of the present invention on CRE-dependent transcriptional activity in rat hippocampal neurons]
The primary hippocampal neurons described above were dispersed and purified, seeded in a culture device (48 well plate) at 8 × 10 ⁴ cells / well, cultured at 37 ° C. for 11 days in a 5% CO ₂ atmosphere, then Lipofect Amine 2000 ( PCRE and Renilla pRG-TK internal standard plasmids were introduced into the cells using Invitrogen) and cultured for 16 hours. Thereafter, the cells were collected after 8 hours of drug stimulation, and a cAMP response element (CRE) that was switched on when the cAMP / PAK / CREB pathway was activated using the Dual-Luciferase Reporter Assy System (Promega). The genes possessed were measured, and thereby the degree of CRE-dependent transcription activity was determined. A representative example of the result is shown in FIG.

  According to the results, when the control group in which cells were treated with a cell culture solution added with DMSO to a final concentration of 0.2% dimethyl sulfoxide (DMSO) is 1, the cell culture solution added with the test substance is used. Was dose-dependent, and approximately 20 times (3.14 ± 0.35) of transcriptional activity was observed at 20 μg / ml, confirming the CRE-dependent transcriptional activity-enhancing effect showing almost the maximum value (saturation).

  In addition, it was confirmed that the medicinal effect of the substance of the present invention is not inferior to that of cells treated with a culture solution added with Forskolin known as a drug that activates adenylate cyclase, a biosynthetic enzyme of cAMP.

[Verification of the enhancement effect of the substance of the present invention on ERK / CREB phosphorylation in rat hippocampal neurons]
In the same manner as above, primary hippocampal neurons were seeded in a 48-well plate at 1 × 10 ⁶ cells / well, cultured at 37 ° C. for 11 days in a 5% CO ₂ atmosphere, followed by drug treatment for 10 minutes. The cells were lysed with Lysis buffer (1 mM EDTA, 1% SDS, 10 mM NaF, 10 nM calyculin, 320 nM okadaic acid, 1 nM sodium orthovanadate, 1 mM p-APMSF, 10 μg / mL antipain, 10 μg / mL leuoeptin, 10 μg / mL chymostain, 10 μg / mL phosphoramidon). 10 μg / mL HEPES pH 7.5), which was heated at 95 ° C. for 10 minutes and then sonicated to obtain a cell lysate. The supernatant obtained by centrifugation (14,000 rpm / 20 min) was converted into SDS, and then the protein was separated on a 12.5% polyacrylamide gel and transferred to a PVDF membrane using SDS-PAGE. The PVDF membrane was blocked with 5% skim milk, and then component search was performed using phosphorylated CREB antibody and phosphorylated ERK antibody (manufactured by Cell Signaling). A representative example of the results of two measurements on this is shown in FIG.

  As a result, by adding the test substance to the cell culture medium, a 3 to 5-fold increase in phosphorylated ERK and phosphorylated CREB was observed in a dose-dependent manner with addition of 10 to 40 μg / ml. Thereby, the enhancement effect of phosphorylation of ERK and CREB by the test substance was confirmed.

[Verification of effects of various signal inhibitors on the enhancement effect of the substance of the present invention on CRE-dependent transcriptional activity in rat hippocampal neurons]
In the same manner as above, primary hippocampal neurons were seeded in a 48-well plate at 8 × 10 ⁴ cells / well, cultured in a 5% CO ₂ atmosphere at 37 ° C. for 11 days, introduced with a plasmid, and cultured for 16 hours. Here, as various signal inhibitors, calmodulin kinase II (CaMKII) inhibitor 3 μM KN62, PKA inhibitor 5 μM H89, and MAP kinase 1/2 (MEK1 / 2) inhibitor 10 μM U0126 after 1 hour pretreatment 8 hours of drug stimulation was applied.

  The cells were collected and measured four times using the Dual-Luciferase Reporter Assay System. A typical example is shown in FIG.

  As a result, by adding 40 μg / ml of the test substance to the cell culture medium, the CRE-dependent transcription activity was about 2 (1.95 ± 0.10), assuming that 1 in the Control group.

On the other hand, the CRE-dependent transcriptional activity in the presence of the above concentrations of KN62, H89 and U0126 is 1.47 ± 0.08, 1.26 ± 0.02, and 1. 1.18 ± 0.08. That is, it has been clarified that the enhancement effect is suppressed by an inhibitor of a signal transduction system (CaMKII, PKA, MEK1 / 2) activated by CREB. In other words, this proves that the mechanism of action of the present invention is the activation of CREB.

  As described above, as a result of examining the improvement effect of memory learning disorder on 24 kinds of citrus whole fruit homogenous liquids, citrus whole fruit homogenous liquids exhibited phosphorylation of CREB (enhancement of CRE transcriptional activity). It has been shown that memory impairment can be improved by promoting and activating the signal transduction system mediated by CREB. At this time, the optimal concentration of the whole fruit homogenate was 20 to 40 μg / ml. From this result, it was also found that by applying the citrus whole fruit homogenate according to the present invention to a known formulation technique, it is possible to provide a drug that produces an effect of improving learning memory impairment.

  The pulverized juice of all citrus fruits according to the present invention and the extract from the crushed juice are applicable to functional foods. This functional food refers to foods that are manufactured to fully exert physiological functions (tertiary functions) such as regulation of biological functions. For example, “Health & Nutrition Food Advisory Staff Textbook” (As of July 30, 2003, published by Daiichi Publishing Co., Ltd., pages 92, 93), it is defined as something different from so-called health foods as well as general foods. It is a thing.

  In such a functional food, the blending ratio of the substance of the present invention, which is an active ingredient, to the carrier component is in the range of 5 to 85% by weight, particularly preferably in the range of 30 to 60% by weight.

It is a graph which shows the effect of this invention substance with respect to a learning memory impairment model animal. It is an evaluation graph of the enhancement effect of the substance of the present invention on CRE-dependent transcriptional activity in rat hippocampal neurons. It is an evaluation graph of the enhancement effect of the substance of the present invention on ERK / CREB phosphorylation in rat hippocampal neurons. It is an evaluation graph of the influence of various signal inhibitors with respect to the enhancement effect of the substance of the present invention on CRE-dependent transcriptional activity in rat hippocampal neurons.

Claims (2)

  A functional food for improving learning and memory impairment, comprising at least one of a crushed juice of all citrus fruits and an extract from the crushed juice, characterized by activation of CREB.   The citrus fruits are Hanayu, Korai Tachibana, Shikikitsu, Pheasant, Natsumikan, Daidai, Ponkan, Mediterranean Mandarin, Dancy Tangerine, Obenimikan, Tachibana, Kobenimikan, Nucleated Kishuu, Sikhwasa, Fukurimikan, Capphi, Ota Ponkan, Era The functional food for improving learning and memory impairment according to claim 1, wherein the functional food is selected from citrus fruits including Cleopatra, Koji, Giri Mikan, and Yichang Lemon.

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